Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

Abstract

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.

The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation

“…Previous work has shown caveolin-1 overexpression increases the number of subplasmalemmal vesicles, and is some instances can generate few attached caveolae (23,(25)(26)(27)(28)40). Thus, caveolin-2 seems to serve an important accessory role by enhancing the formation of invaginated caveolae in the presence of caveolin-1 consistent with its role in caveolae formation in various cell types examined by us or others (29,30).…”

“…Ectopic expression of caveolin-1 induces de novo formation of large and small noncoated vesicles that do not fuse significantly with the plasma membrane in cells that normally lack these organelles (23,(25)(26)(27)(28). Evidence supporting the interdependence on both caveolins for the assembly of caveolae stems from studies showing that the interaction of caveolin-1 with caveolin-2 renders caveolin-2 detergent insoluble, targets Golgi localized caveolin-2 to the plasma membrane, and stabilizes caveolin-2 levels in cells.…”

Distinction between signaling mechanisms in lipid rafts vs. caveolae

“…In these cells, overexpression of cav-1 stimulates caveolae biogenesis (26) and the redistribution of cav-2 from the Golgi to Triton X-100 insoluble lipid-rich domains of plasma membrane (21). As seen in Table 1, parental FRT cells did not have a single plasmalemma attached caveolae but contained ample uncoated vesicles, clathrin-coated pits, and vesicles based on quantitative EM.…”

Unravelling protein sorting

“…For example, the epithelial cell line FRT lacks morphological caveolae, its main structural component caveolin and mis-sorts some GPI-anchored proteins to the basolateral membrane. However, although transfection of caveolin 1 promotes the assembly of basolateral caveolae, it does not restore correct sorting of GPI-anchored proteins in these cells 11 . Furthermore, it is not yet clear whether caveolae facilitate or retard the internalization of GPI-anchored proteins 12 .…”